Heterocyclic inhibitors of necroptosis

ABSTRACT

The invention features a series of heterocyclic derivatives that inhibit tumor necrosis factor alpha (TNF-α) induced necroptosis. The heterocyclic compounds of the invention are described by Formulas (I) and (Ia)-(Ie) and are shown to inhibit TNF-α induced necroptosis in FADD-deficient variant of human Jurkat T cells. The invention further features pharmaceutical compositions featuring the compounds of the invention. The compounds and compositions of the invention may also be used to treat disorders where necroptosis is likely to play a substantial role.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of application Ser. No. 12/228,750,filed Aug. 15, 2008, which claims benefit of U.S. ProvisionalApplication No. 60/955,966, filed Aug. 15, 2007, and 61/038,175, filedMar. 20, 2008, each of which is hereby incorporated by reference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

The present research was supported by grants from the NationalInstitutes of Health (Grant No. GM-64703 and Grant No. U01 NS050560).The U.S. government has certain rights to this invention.

FIELD OF THE INVENTION

The invention relates to heterocyclic compounds and to cell death, inparticular through necrosis and necroptosis, and regulation thereof byheterocyclic compounds.

BACKGROUND OF THE INVENTION

In many diseases, cell death is mediated through apoptotic and/ornecrotic pathways. While much is known about the mechanisms of actionthat control apoptosis, control of necrosis is not as well understood.Understanding the mechanisms regulating both necrosis and apoptosis incells is essential to being able to treat conditions, such asneurodegenerative diseases, stroke-coronary heart disease, kidneydisease, and liver disease. A thorough understanding of necrotic andapoptotic cell death pathways is also crucial to treating AIDS and theconditions associated with AIDS, such as retinal necrosis.

Cell death has traditionally been categorized as either apoptotic ornecrotic based on morphological characteristics (Wyllie et al., Int.Rev. Cytol. 68: 251 (1980)). These two modes of cell death were alsoinitially thought to occur via regulated (caspase-dependent) andnon-regulated processes, respectively. More recent studies, however,demonstrate that the underlying cell death mechanisms resulting in thesetwo phenotypes are much more complicated and under some circumstancesinterrelated. Furthermore, conditions that lead to necrosis can occur byeither regulated caspase-independent or non-regulated processes.

One regulated caspase-independent cell death pathway with morphologicalfeatures resembling necrosis, called necroptosis, has recently beendescribed (Degterev et al., Nat. Chem. Biol. 1:112 (2005)). This mannerof cell death can be initiated with various stimuli (e.g., TNF-α and Fasligand) and in an array of cell types (e.g., monocytes, fibroblasts,lymphocytes, macrophages, epithelial cells and neurons). Necroptosis mayrepresent a significant contributor to and in some cases predominantmode of cellular demise under pathological conditions involvingexcessive cell stress, rapid energy loss and massive oxidative speciesgeneration, where the highly energy-dependent apoptosis process is notoperative.

The identification and optimization of low molecular weight moleculescapable of inhibiting necroptosis will assist in elucidating its role indisease patho-physiology and could provide compounds (i.e.,necrostatins) for anti-necroptosis therapeutics. The discovery ofcompounds that prevent caspase-independent cell death (e.g., necrosis ornecroptosis) would also provide useful therapeutic agents for treatingor preventing conditions in which necrosis occurs. These compounds andmethods would be particularly useful for the treatment ofneurodegenerative diseases, ischemic brain and heart injuries, and headtrauma.

SUMMARY OF THE INVENTION

In one aspect, the invention features a compound having a structureaccording to Formula (I)

wherein

X₁ and X₂ are, independently, N or CR⁴;

X₃ is selected from O, S, NR⁵, or —(CR⁵)₂;

Y is selected from C(O) or CH₂; and

Z is (CR⁶R⁷)_(n),

R¹ is selected from H, halogen, optionally substituted C₁₋₆ lower alkyl,or optionally substituted C₁₋₆ cycloalkyl, or optionally substitutedaryl;

R² is selected from H or optionally substituted C₁₋₆ lower alkyl;

R³ is optionally substituted aryl;

each R⁴ is selected from H, halogen, carboxamido, nitro, cyano,optionally substituted lower C₁₋₆ alkyl, or optionally substituted aryl;

R⁵ is selected from H, halogen, optionally substituted lower C₁₋₆ alkyl,or optionally substituted aryl;

each R⁶ and R⁷ is, independently, selected from H, optionallysubstituted aryl, or optionally substituted C₁₋₆ lower alkyl; and

n is 0, 1, 2, or 3;

where

when X₁ and X₂ are N, X₃ is S, Y, is C(O), Z is CH₂, R² is H, and R³ is2-chloro-6-fluoro-phenyl, R¹ is not methyl;

or any pharmaceutically acceptable salt or solvate thereof, orstereoisomer thereof.

In some embodiments, both X₁ and X₂ are N or both X₁ and X₂ are CR⁴,wherein X₃ is not NR⁵ when X₁ and X₂ are N.

In some embodiments, X₁ and X₂ are N and X₃ is S.

In some embodiments, X₁ and X₂ are CR⁴ and X₃ is NR⁵.

In some embodiments, R¹ is C₁₋₆ cycloalkyl or branched C₁₋₆ lower alkyl.

In some embodiments R² is H.

In some embodiments, R³ is a substituted phenyl group having thestructure

where

each R⁸, R⁹, R¹⁰, R¹¹, and R¹² is selected, independently, from H, lowerC₁₋₆ alkyl, halogen, amino, carboxamido, alkoxy, nitro, and cyano, andat least one of R⁸, R⁹, R¹⁰, R¹¹, and R¹² is not hydrogen.

In some embodiments, R⁸ and and R¹² are, independently, halogen. Inother embodiments, R⁸ and and R¹² are, independently, fluorine orchlorine. In certain embodiments, R⁸ is fluorine and R¹² is chlorine.

In some embodiments, R⁹, R¹⁰, and R¹¹ are hydrogen.

In some embodiments, R¹ is C₁₋₆ cycloalkyl or branched C₁₋₆ lower alkyl,or any pharmaceutically acceptable salt or solvate thereof, orstereoisomer thereof. In other embodiments, the compound of Formula (I)has a structure according to Formula (I-a)

where R¹, R², R³, R⁶, and R⁷ are as defined for Formula (I), or anypharmaceutically acceptable salt or solvate thereof, or stereoisomerthereof.

In some embodiments, R³ is a substituted phenyl group having thestructure

where each R⁸, R⁹, R¹⁰, R¹¹, and R¹² is selected, independently, from H,lower C₁₋₆ alkyl, halogen, amino, carboxamido, alkoxy, nitro, and cyano,and at least one of R⁸, R⁹, R¹⁰, R¹¹, and R¹² is not hydrogen.

In some embodiments, R⁸ and and R¹² are, independently, halogen. Inother embodiments, R⁸ and and R¹² are, independently, fluorine orchlorine. In certain embodiments, R⁸ is fluorine and R¹² is chlorine.

In some embodiments, R⁹, R¹⁰, and R¹¹ are hydrogen, or anypharmaceutically acceptable salt or solvate thereof, or stereoisomerthereof.

In some embodiments, R¹ is C₁₋₆ cycloalkyl or branched C₁₋₆ lower alkyl,or any pharmaceutically acceptable salt or solvate thereof, orstereoisomer thereof. In certain embodiments, R¹ is cyclopropyl,cyclobutyl, or isopropyl.

In some embodiments, R² is H, or any pharmaceutically acceptable salt orsolvate thereof, or stereoisomer thereof.

In some embodiments, R⁶ and R⁷ are both hydrogen, or anypharmaceutically acceptable salt or solvate thereof, or stereoisomerthereof.

In some embodiments, R⁶ is hydrogen and R⁷ is lower C₁₋₆ alkyl, or anypharmaceutically acceptable salt or solvate thereof, or stereoisomerthereof. In certain embodiments, carbon bearing R⁶ and R⁷ has the(S)-configuration.

In other embodiments, the compound of Formula (I) has a structureaccording to Formula (I-b)

where Y and Z are as defined for Formula (I) and R is selected from:hydrogen, halogen, azido, cyano, nitro, optionally substituted lowerC₁₋₆ alkyl, aryl, alkoxy, aryloxy, amino, carboxylic group, ketone,carbonate, ester, carboxamide, or carbamate, or any pharmaceuticallyacceptable salt or solvate thereof, or stereoisomer thereof.

In some embodiments, Y is C(O). In other embodiments, Y is CH₂.

In some embodiments, R is halogen. In certain embodiments, R ischlorine, or any pharmaceutically acceptable salt or solvate thereof, orstereoisomer thereof.

In some embodiments, Z is CH₂. In other embodiments, Z is CHR⁷, where R⁷is C₁₋₆ lower alkyl. In certain embodiments, the carbon bearing R⁷ hasthe S-configuration.

In some embodiments, the compound having a structure according to (I-b)is

or any pharmaceutically acceptable salt or solvate thereof, orstereoisomer thereof.

In other embodiments, the compound of Formula (I) has a structureaccording to Formula (I-c)

wherein

R¹, R², and R⁷ are as defined for Formula (I);

R^(4A) and R^(4B) are selected, independently, from hydrogen, halogen,carboxamido, nitro, and cyano;

R⁵ is H or optionally substituted C₁₋₆ lower alkyl;

each of R⁸, R⁹, R¹⁰, R¹¹, and R¹² is selected, independently, from H,lower C₁₋₆ alkyl, halogen, amino, amido, alkoxy, nitro, and cyano, andat least one of R⁸, R⁹, R¹⁰, R¹¹, and R¹² is not hydrogen;

or any pharmaceutically acceptable salt or solvate thereof, orstereoisomer thereof

In some embodiments, R¹ is H.

In some embodiments, R² is H.

In some embodiments, R^(4A) is H and R^(4B) is CN. In other embodiments,R^(4A) is CN and R^(4B) is H.

In some embodiments, R⁵ is unsubstituted C₁₋₆ lower alkyl.

In some embodiments, R⁷ is C₁₋₆ lower alkyl. In other embodiments, thecarbon bearing R⁷ has the S-configuration.

In some embodiments, R⁸ and R¹² are each, independently, halogen.

In some embodiments, R⁹, R¹⁰, and R¹¹ are hydrogen.

In other embodiments, the compound of Formula (I) has a structureaccording to Formula (I-d)

wherein R^(4B), R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹¹ and R¹² are as defined inFormula (I-c), or any pharmaceutically acceptable salt or solvatethereof, or stereoisomer thereof.

In some embodiments, R^(4B) is CN.

In some embodiments, R⁵ is unsubstituted C₁₋₆ lower alkyl.

In some embodiments, R⁷ is C₁₋₆ lower alkyl. In certain embodiments, thecarbon bearing R⁷ has the S-configuration.

In some embodiments, R⁸ and R¹² are each, independently, halogen.

In some embodiments, R⁹, R¹⁰, and R¹¹ are hydrogen.

In other embodiments, the compound of Formula (I) has a structureaccording to Formula (I-e)

wherein R¹, R², R^(4A), R^(4B), R⁵, and R⁷ are as defined in Formula(I-c), or any pharmaceutically acceptable salt or solvate thereof, orstereoisomer thereof.

In some embodiments, R¹ is H or unsubstituted C₁₋₆ lower alkyl.

In some embodiments, R² is H.

In some embodiments, R^(4A) is H and R^(4B) is CN.

In some embodiments, R^(4A) is CN and R^(4B) is H.

In some embodiments, R⁷ is hydrogen.

In some embodiments, R⁷ is C₁₋₆ lower alkyl. In certain embodiments, thecarbon bearing R⁷ has the S-configuration, or any pharmaceuticallyacceptable salt or solvate thereof, or stereoisomer thereof.

In some embodiments, the compound of Formula (I-e) is

In some embodiments, the compound of Formula (I) is selected from thegroup consisting of:

or any pharmaceutically acceptable salt or solvate thereof, orstereoisomer thereof.

The compounds of Formulas (I), (I-a), (I-b), (I-c), (I-d), or (I-e) alsoinclude any pharmaceutically acceptable salts or solvates thereof, orstereoisomers thereof.

In a second aspect, the invention features a pharmaceutical compositionincluding a pharmaceutically acceptable excipient and the compound ofany of Formulas (I), (I-a), (I-b), (I-c), (I-d), or (I-e), or thecompound having the formula:

or any pharmaceutically acceptable salt or solvate thereof, orstereoisomer thereof.

In a third aspect, the invention features a method of treating acondition in a subject, where the method includes the step ofadministering the compound of any of Formulas (I), (I-a), (I-b), (I-c),(I-d), or (I-e), or the compound having the formula:

or any pharmaceutically acceptable salt or solvate thereof, orstereoisomer thereof, to said subject in a dosage sufficient to decreasenecroptosis. In some embodiments, the condition is a neurodegenerativedisease or is caused by alteration in cell proliferation,differentiation, or intracellular signalling.

In a fourth aspect, the invention features a method of decreasingnecroptosis including the step of contacting a cell with the compound ofany of Formulas (I), (I-a), (I-b), (I-c), (I-d), or (I-e), or thecompound having the formula:

or any pharmaceutically acceptable salt or solvate thereof, orstereoisomer thereof. In some embodiments, the condition is aneurodegenerative disease or is a condition caused by alteration in cellproliferation, differentiation, or intracellular signalling. In someembodiments, the condition caused by alteration in cell proliferation,differentiation, or intracellular signalling is cancer or infection(e.g., by viruses (e.g., acute, latent and persistent), bacteria, fungi,or other microbes). In some embodiments, the viruses are humanimmunodeficiency virus (HIV), Epstein-Barr virus (EBV), cytomegalovirus(CMV)5 human herpesviruses (HHV), herpes simplex viruses (HSV), humanT-Cell leukemia viruses (HTLV)5 Varicclla-Zoster virus (VZV), measlesvirus, papovaviruses (JC and BK), hepatitis viruses, adenovirus,parvoviruses, and human papillomaviruses. In other embodiments, thecondition is a neurodegenerative disease that is Alzheimer's disease,Huntington's disease, Parkinson's disease, amyotrophic lateralsclerosis, HIV-associated dementia, cerebral ischemia, amyotropiclateral sclerosis, multiple sclerosis, Lewy body disease, Menke'sdisease, Wilson's disease, Creutzfeldt-Jakob disease, Fahr disease, ormuscular dystrophies or related diseases (e.g., Becker's musculardystrophy, Duchenne muscular dystrophy, myotonic dystrophy, limb-girdlemuscular dystrophy, Landouzy-Dejerine muscular dystrophy,facioscapulohumeral muscular dystrophy (Steinert's disease), myotoniacongenita, Thomsen's disease, and Pompe's disease). In some embodiments,the neurodegerative disease is muscle wasting. In other embodiments,muscle wasting is associated with cancer, AIDS, congestive heartfailure, chronic obstructive pulmonary disease, and necrotizing myopathyof intensive care.

In a fifth aspect, the invention features a method of screeningcompounds to identify inhibitors of necroptosis, where the methodincludes the following steps:

-   -   (a) providing a first cell in which necroptosis is inhibited;    -   (b) contacting the cell of (a) with a compound that inhibits        necroptosis;    -   (c) comparing the inhibition of necroptosis observed in (b) to        the inhibition of necroptosis observed by contacting a cell with        the compound of any of Formulas (I), (I-a), (I-b), (I-c), (I-d),        or (I-e), or the compound having the formula:

-   -   or any pharmaceutically acceptable salt or solvate thereof, or        stereoisomer thereof,

wherein an inhibitor of necroptosis is identified when the inhibition ofnecroptosis observed in (b) exceeds the inhibition of necroptosis by thecompound of any of Formulas (I), (I-a), (I-b), (I-c), (I-d), or (I-e),or any pharmaceutically acceptable salt or solvate thereof, orstereoisomer thereof.

In a sixth aspect, the invention features a kit including

-   -   (a) a pharmaceutically acceptable composition that includes the        compound of any of Formulas (I), (I-a), (I-b), (I-c), (I-d), or        (I-e), or the compound having the formula:

-   -   or any pharmaceutically acceptable salt or solvate thereof, or        stereoisomer thereof; and    -   (b) instructions for the use of the pharmaceutical composition        of (a) to treat a condition in a subject.

In another aspect, the compounds of Formulas (I), (I-a), (I-b), (I-d),or (I-e) are used to boost the immune system in a patient. In someembodiments, the patient has an immunocompromising condition. In otherembodiments, the patient does not have an immunocompromising condition.

By “alkoxy” is meant a group having the structure —O(lower C₁₋₆ alkyl),where the lower C₁₋₆ alkyl may be brached, linear, or cyclic. The lowerC₁₋₆ alkyl may also be substituted or unsubstituted.

By “amino” is meant a group having a structure selected from: —NH₂,—NH(lower C₁₋₆ alkyl), —N(lower C₁₋₆ alkyl)₂, —NH(aryl), —N(lower C₁₋₆alkyl)(aryl), and —N(aryl)₂. Each lower C₁₋₆ alkyl and aryl may be,independently, unsubstituted or substituted. Each lower C₁₋₆ alkyl maybe, independently, brached, linear, or cyclic.

By “aryl” is meant is an optionally substituted C₆-C₁₄ cyclic group with[4n+2] π electrons in conjugation and where n is 1, 2, or 3.Non-limiting examples of arenes include heteroaryls and benzene,naphthalene, anthracene, and phenanthrene. Aryls may be unsubstituted orsubstituted. A substituted aryl may be optionally substituted with 1, 2,3, 4, 5, or 6 substituents located at any position of the ring.

By “aryloxy” is meant a group having the structure —O(aryl). Aryl may beunsubstituted or substituted.

By “azido” is meant a group having the structure —N₃.

By “carbamate” is meant a group having the structure —OCONH₂,—OCONH(lower C₁₋₆ alkyl), —OCON(lower C₁₋₆ alkyl)₂, —OCON(lower C₁₋₆alkyl)(aryl), —OCONH(aryl), or —OCON(aryl)₂. Each lower C₁₋₆ alkyl andaryl may be, independently, unsubstituted or substituted. Each lowerC₁₋₆ alkyl may be, independently, brached, linear, or cyclic.

By “carbonate” is meant a group having a the structure —OCO₂(lower C₁₋₆alkyl) or —OCO₂(aryl). Each lower C₁₋₆ alkyl and aryl may beunsubstituted or substituted.

By “carboxamide” is meant a group having the structure —CONH₂,—CON(lower C₁₋₆ alkyl), —CON(lower C₁₋₆ alkyl)₂, —CON(lower C₁₋₆alkyl)(aryl), —CONH(aryl), or —CON(aryl)₂. Each lower C₁₋₆ alkyl andaryl may be, independently, unsubstituted or substituted. Each lowerC₁₋₆ alkyl may be, independently, brached, linear, or cyclic.

By “carboxylic group” is meant a group having a structure selected from:—CO₂H, —CO₂(lower C₁₋₆ alkyl), and —CO₂(aryl). Each lower C₁₋₆ alkyl andaryl may be unsubstituted or substituted. Each lower C₁₋₆ alkyl may be,independently, brached, linear, or cyclic.

By “cyano” is meant a group having the structure —CN.

By “effective amount” or “therapeutically effective amount” of an agent,as used herein, is that amount sufficient to effect beneficial ordesired results, such as clinical results, and, as such, an effectiveamount depends upon the context in which it is being applied. Forexample, in the context of administering an agent that is an inhibitorof necroptosis, an effective amount of an agent is, for example, anamount sufficient to achieve a reduction in necroptosis as compared tothe response obtained without administration of the agent.

By “ester” is meant a group having a structure selected from —OCO(lowerC₁₋₆ alkyl) or —OCO(aryl). Each lower C₁₋₆ alkyl and aryl may beunsubstituted or substituted. Each lower C₁₋₆ alkyl may be,independently, brached, linear, or cyclic.

By “halogen” or “halo” is meant fluorine (—F), chlorine (—Cl), bromine(—Br), or iodine (—I).

By “heteroaryl” is mean an aryl group that contains 1, 2, or 3heteroatoms in the cyclic framework. Exemplary heteroaryls include, butare not limited to, furan, thiophene, pyrrole, thiadiazole (e.g.,1,2,3-thiadiazole or 1,2,4-thiadiazole), oxadiazole (e.g.,1,2,3-oxadiazole or 1,2,5-oxadiazole), oxazole, benzoxazole, isoxazole,isothiazole, pyrazole, thiazole, benzthiazole, triazole (e.g.,1,2,4-triazole or 1,2,3-triazole), benzotriazole, pyridines,pyrimidines, pyrazines, quinoline, isoquinoline, purine, pyrazine,pteridine, triazine (e.g, 1,2,3-triazine, 1,2,4-triazine, or1,3,5-triazine)indoles, 1,2,4,5-tetrazine, benzo[b]thiophene,benzo[c]thiophene, benzofuran, isobenzofuran, and benzimidazole.Heteroaryls may be unsubstituted or substituted with 1, 2, 3, 4, 5, or 6subsitutents.

By “ketone” is meant a group having the structure —CO(lower C₁₋₆ alkyl)or —CO(aryl). Each lower C₁₋₆ alkyl and aryl may be unsubstituted orsubstituted. Each lower C₁₋₆ alkyl may be, independently, brached,linear, or cyclic.

By “lower alkyl” or “lower C₁₋₆ alkyl” is meant hydrocarbon chains offrom 1 to 6 carbon atoms. Lower alkyls may include 1, 2, 3, 4, 5, or 6carbon atoms. A lower C₁₋₆ alkyl may be linear, branched or cyclic(“cycloalkyls”). Examples of linear lower alkyl groups include, but arenot limited to: methyl, ethyl, n-propyl, n-butyl, n-pentyl, and n-hexyl.Examples of branched lower alkyl groups include, but are not limited to:isopropyl, s-, i- and t-butyl, and isoamyl. Examples of cyclic loweralkyl groups include, but are not limited to: cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, and cyclobutylmethyl. Desirably, a lower C₁₋₆alkyl is methyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, orcyclopropyl. A lower alkyl may be unsubstituted or substituted. Asubstituted lower alkyl may be optionally substituted with 1, 2, 3, 4,5, or 6 substituents located at any carbon of the lower alkyl.

By “nitro” is meant a group having the structure —NO₂.

A “pharmaceutically acceptable excipient” as used herein refers anyingredient other than the compounds described herein (for example, avehicle capable of suspending or dissolving the active compound) andhaving the properties of being nontoxic and non-inflammatory in apatient. Excipients may include, for example: antiadherents,antioxidants, binders, coatings, compression aids, disintegrants, dyes(colors), emollients, emulsifiers, fillers (diluents), film formers orcoatings, flavors, fragrances, glidants (flow enhancers), lubricants,preservatives, printing inks, sorbents, suspensing or dispersing agents,sweeteners, or waters of hydration. Exemplary excipients include, butare not limited to: butylated hydroxytoluene (BHT), calcium carbonate,calcium phosphate (dibasic), calcium stearate, croscarmellose,crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine,ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol,methionine, methylcellulose, methyl paraben, microcrystalline cellulose,polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinizedstarch, propyl paraben, retinyl palmitate, shellac, silicon dioxide,sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate,sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc,titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

The term “pharmaceutically acceptable salt,” as used herein, representsthose salts which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of humans and animalswithout undue toxicity, irritation, allergic response and the like andare commensurate with a reasonable benefit/risk ratio. Pharmaceuticallyacceptable salts are well known in the art. For example, S. M. Berge etal. describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences, 1977, 66:1-19. The salts can be prepared insitu during the final isolation and purification of the compounds of theinvention or separately by reacting the free base group with a suitableorganic acid. Representative acid addition salts include acetate,adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate,bisulfate, borate, butyrate, camphorate, camphersulfonate, citrate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate,hexanoate, hydrobromide, hydrochloride, hydroiodide,2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, laurylsulfate, malate, maleate, malonate, methanesulfonate,2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate,pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, stearate, succinate, sulfate, tartrate,thiocyanate, toluenesulfonate, undecanoate, valerate salts and the like.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium and the like, as well as nontoxicammonium, quaternary ammonium, and amine cations, including, but notlimited to ammonium, tetramethylammonium, tetraethylammonium,methylamine, dimethylamine, trimethylamine, triethylamine, ethylamineand the like.

The term “pharmaceutically acceptable solvates,” as used herein, refersto compounds that retain non-covalent associations to residual solventmolecules in the solid state. For example, solvates may be prepared bycrystallization, recrystallization, or precipitation from a solutionthat includes organic solvents, water, or a mixture thereof. Solvatesinclude, but are not limited to, compounds that include solventmolecules in the crystal lattice following recrystallization. Themolecular stoichiometry of solvation can vary from, for example, 1:1solvent:compound to 10:1 solvent:compound. These ratios can include amixture of associated solvent molecules. Exemplary, non-limitingexamples of solvents that can form solvates with the compounds of theinvention include water (for example, mono-, di-, and tri-hydrates),N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO),N,N′-dimethylformamide (DMF), N,N′-dimethylacetamide (DMAC),1,3-dimethyl-2-imidazolidinone (DMEU),1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile(ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-pyrrolidone,benzyl benzoate, or any combination thereof.

By “pharmaceutical composition” is meant a composition containing acompound of the invention, formulated with a pharmaceutically acceptableexcipient, and manufactured or sold with the approval of a governmentalregulatory agency as part of a therapeutic regimen for the treatment ofdisease in a mammal. Excipients consisting of DMSO are specificallyexcluded. Pharmaceutical compositions can be formulated, for example,for oral administration in unit dosage form (e.g., a tablet, capsule,caplet, gelcap, or syrup); for topical administration (e.g., as a cream,gel, lotion, or ointment); for intravenous administration (e.g., as asterile solution free of particulate emboli and in a solvent systemsuitable for intravenous use); or any other formulation describedherein.

By “stereoisomer” is meant a diastereomer, enantiomer, or epimer of acompound. A chiral center in a compound may have the S-configuration orthe R-configuration. Diastereomers of a compound include stereoisomersin which some, but not all, of the chiral centers have the oppositeconfiguration as well as those compounds in which substituents aredifferently oriented in space (for example, trans versus cis).

Where a group is substituted, the group may be substituted with 1, 2, 3,4, 5, or 6 substituents. Optional substituents include, but are notlimited to: halogen, azido, cyano, nitro, lower C₁₋₆ alkyl, aryl,alkoxy, aryloxy, amino, carboxylic group, ketone, carbonate, ester,carboxamide, or carbamate. Substituents may be further substituted with1, 2, 3, 4, 5, or 6 substituents as defined herein. For example, a lowerC₁₋₆ alkyl or an aryl group (e.g., heteroaryl, phenyl, or naphthyl) maybe further substituted with 1, 2, 3, 4, 5, or 6 substituents.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows cell-type-specific activities of necrostatins.FADD-deficient Jurkat, L929 and mouse adult lung fibroblast cells weretreated for 24 hours with 10 ng/mL human TNF-α and/or 100 μM zVAD.fmk asindicated in the presence of 30 μM of necrostatin 1, 2 or 55. Cellviability was determined using a commercial cell viability kit. Valueswere normalized to cells treated with necrostatins in the absence ofnecroptotic stimulus, which were set as 100% viability. Error barsreflect standard deviation values (N=2).

DETAILED DESCRIPTION OF THE INVENTION

We have discovered a series of heterocyclic derivatives that inhibittumor necrosis factor alpha (TNF-α)-induced necroptosis. Theheterocyclic compounds of the invention are described by Formulas (I)and (Ia)-(Ie) and are shown to inhibit TNF-α induced necroptosis inFADD-deficient variant of human Jurkat T cells. Pharmaceuticalcompositions including the compounds of the invention are alsodescribed. The invention also features kits and methods of treatmentfeaturing the compounds and compositions of the invention.

Compounds of the invention are described generally by Formula (I):

wherein

X₁ and X₂ are, independently, N or CR⁴;

X₃ is selected from O, S, NR⁵, or —(CR⁵)₂;

Y is selected from C(O) or CH₂; and

Z is (CR⁶R⁷)_(n),

R¹ is selected from H, halogen, optionally substituted C₁₋₆ lower alkyl,or optionally substituted C₁₋₆ cycloalkyl;

R² is selected from H or optionally substituted C₁₋₆ lower alkyl;

R³ is optionally substituted aryl;

each R⁴ is selected from H, halogen, optionally substituted lower C₁₋₆alkyl, or optionally substituted aryl;

R⁵ is selected from H, halogen, optionally substituted lower C₁₋₆ alkyl,or optionally substituted aryl;

each R⁶ and R⁷ is, independently, selected from H, optionallysubstituted aryl, or optionally substituted C₁₋₆ lower alkyl; and

n is 0, 1, 2, or 3; where

when X₁ and X₂ are N, X₃ is S, Y, is C(O), Z is CH₂, R² is H, and R³ is2-chloro-6-fluoro-phenyl, R¹ is not methyl;

or any pharmaceutically acceptable salt or solvate thereof, orstereoisomer thereof.

In one embodiment, the compounds of the invention have the Formula(I-a):

where R¹ is selected from H, halogen, optionally substituted C₁₋₆ loweralkyl, optionally substituted C₁₋₆ cycloalkyl, or optionally substitutedaryl;

R² is selected from H or optionally substituted C₁₋₆ lower alkyl;

R³ is optionally substituted aryl;

R⁶ and R⁷ are, independently, selected from H or optionally substitutedC₁₋₆ lower alkyl; and

at least one of R⁶ and R⁷ is hydrogen.

Compounds of the invention having Formula (I-a) include:

Compounds of the invention also include those having Formula (I-b):

wherein Y and Z are as defined for Formula (I) and R is a substituentthat may be selected from: hydrogen, halogen, azido, cyano, nitro,optionally substituted lower C₁₋₆ alkyl, aryl, alkoxy, aryloxy, amino,carboxylic group, ketone, carbonate, ester, carboxamide, or carbamate.

Compounds of the invention having Formula (I-b) include:

Compounds of the invention can also have a structure according toFormula (I-c):

where

R¹ is selected from H, halogen, optionally substituted C₁₋₆ lower alkyl,optionally substituted C₁₋₆ cycloalkyl, or optionally substituted aryl;

R² is selected from H or optionally substituted C₁₋₆ lower alkyl;

R^(4A) and R^(4B) are selected, independently, from hydrogen, halogen,carboxamido, nitro, and cyano;

R⁵ is H or optionally substituted C₁₋₆ lower alkyl;

R⁷ is hydrogen or optionally substituted lower C₁₋₆ alkyl;

each of R⁸, R⁹, R¹⁰, R¹¹, and R¹² is selected, independently, from H,lower C₁₋₆ alkyl, halogen, amino, amido, alkoxy, nitro, and cyano; and

at least one of R⁸, R⁹, R¹⁰, R¹¹, and R¹² is not hydrogen.

Examples of compounds having Formula (I-c) include those having Formula(I-d):

where R^(4B), R⁵, R⁷, R⁸, R⁹, R¹⁰, R¹¹, and R¹² are as defined inFormula (I-c).

Additional examples of compounds having Formula (I-c) include thosehaving Formula (I-e):

where R¹, R², R^(4A), R^(4B), R⁵, and R⁷ are as defined in Formula(I-c).

Based upon these results, the following chemical formula representscompounds according to the invention.

wherein R¹ is a lower alkyl group;

-   -   R² is selected from hydrogen and an electron withdrawing group;    -   R³ is selected from hydrogen and an (S)-lower alkyl group;    -   each of R4, R5 and R6 is independently selected from hydrogen,        lower    -   alkyl, halogen, amino, amido, alkoxy and cyano; and    -   each X¹ and X² is an electron withdrawing group.

In certain preferred embodiments,

-   -   R¹ is selected from methyl, ethyl, propyl and butyl;    -   R² is selected from hydrogen, halogen and cyano;    -   R³ is selected from hydrogen, (S)-methyl, (S)-ethyl, (S)-propyl        and (S)-butyl; and    -   each X¹ and X² is an independent halogen.

In certain preferred embodiments,

-   -   R¹ is selected from methyl and isopropyl;    -   R² is selected from hydrogen, chlorine, bromine and cyano;    -   R³ is selected from hydrogen and (S)-methyl; and    -   each X¹ and X² is independently chlorine or fluorine.

In one particularly preferred embodiment, R¹ is methyl, R² is cyano, R³is (S)-methyl, X¹ is chlorine and X² is fluorine.

In the compounds according to the invention each of R¹, R², R³, X¹ andX² may be independently selected from the substituents and preferredsubstituents disclosed above.

The term “lower alkyl” as employed herein refers to straight andbranched chain aliphatic groups having from 1 to 6 carbon atoms, whichis optionally substituted with one, two or three substituents. Preferredalkyl groups include, without limitation, methyl, ethyl, propyl,isopropyl, butyl, isobutyl, sec-butyl and tert-butyl.

Exemplary compounds of the invention having Formula (I-e) include thosedepicted in Table 1:

TABLE 1 R¹ R² R^(4A) R^(4B) R⁵ R⁷ Structure 78 H H H Cl Me H

80 H H H H H H

81 Me H H H H H

82 Me H H H Me H

83 Me H H H i-Pr H

84 H H H H Me H

85 H H H H i-Pr H

86 H H H H Bn H

87 H H H H CH(Me)Ph H

88 H Me H H Me H

89 H H H H i-Pr (±)-Me

90 H H H H i-Pr (S)-Me

91 H H H H i-Pr (R)-Me

92 H H H H i-Pr (±)-n-Bu

93 H H H Br Et H

94 H H H CN Me H

95 H H H CN Et H

96 H H H CN i-Pr H

97 H H H CN Me (S)-Me

98 H H CN H Me H

99 H H CN H i-Pr H

Compounds of the invention where the carbon bearing R⁷ is a chiralcenter may be used as a racemate, stereochemical mixture, or inenantiomerically pure form. In one embodiment, a compound where wherethe carbon bearing R⁷ is a chiral center has the (S)-configuration. Inanother embodiment, a compound where where the carbon bearing R⁷ is achiral center has the (R)-configuration.

Compounds of the invention can be synthesized according to methods knownin the art or by the methods provided in the examples below. Forexample, compounds of the invention (e.g., compounds of Formula (I-b))may be prepared according to the method illustrated in Scheme 1 andpresented in Example 1.

Pharmaceutical Compositions

The compounds of the invention (e.g., (Formulas (I) and (I-a)-(I-e)) canbe formulated into pharmaceutical compositions for administration tohuman subjects in a biologically compatible form suitable foradministration in vivo. Accordingly, in another aspect, the presentinvention provides a pharmaceutical composition comprising a compound ofthe invention in admixture with a pharmaceutically acceptable excipient.Conventional procedures and ingredients for the selection andpreparation of suitable formulations are described, for example, inRemington's Pharmaceutical Sciences (2003-20^(th) edition) and in TheUnited States Pharmacopeia: The National Formulary (USP 24 NF19),published in 1999.

The compounds of the invention may be used in the form of the free base,in the form of salts, solvates, and as prodrugs. All forms are withinthe scope of the invention. In accordance with the methods of theinvention, the described compounds or salts, solvates, or prodrugsthereof may be administered to a patient in a variety of forms dependingon the selected route of administration, as will be understood by thoseskilled in the art. The compounds of the invention may be administered,for example, by oral, parenteral, buccal, sublingual, nasal, rectal,patch, pump, or transdermal administration and the pharmaceuticalcompositions formulated accordingly. Parenteral administration includesintravenous, intraperitoneal, subcutaneous, intramuscular,transepithelial, nasal, intrapulmonary, intrathecal, rectal, and topicalmodes of administration. Parenteral administration may be by continuousinfusion over a selected period of time.

Pharmaceutically Acceptable Excipients

Pharmaceutically acceptable excipients may include, for example:antiadherents, antioxidants, binders, coatings, compression aids,disintegrants, dyes (colors), emollients, emulsifiers, fillers(diluents), film formers or coatings, flavors, fragrances, glidants(flow enhancers), lubricants, preservatives, printing inks, sorbents,suspensing or dispersing agents, sweeteners, or waters of hydration.Exemplary excipients include, but are not limited to: butylatedhydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic),calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone,citric acid, crospovidone, cysteine, ethylcellulose, gelatin,hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose,magnesium stearate, maltitol, mannitol, methionine, methylcellulose,methyl paraben, microcrystalline cellulose, polyethylene glycol,polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben,retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch(corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide,vitamin A, vitamin E, vitamin C, and xylitol.

Oral Administration

A compound of the invention may be orally administered, for example,with an inert diluent or with an assimilable edible carrier, or it maybe enclosed in hard or soft shell gelatin capsules, or it may becompressed into tablets, or it may be incorporated directly with thefood of the diet. For oral therapeutic administration, a compound of theinvention may be incorporated with an excipient and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like.

Parenteral Administration

A compound of the invention may also be administered parenterally. Thepharmaceutical forms suitable for injectable use include sterile aqueoussolutions or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. In all casesthe form must be sterile and must be fluid to the extent that may beeasily administered via syringe.

Nasal Administration

Compositions for nasal administration may conveniently be formulated asaerosols, drops, gels, and powders. Aerosol formulations typicallyinclude a solution or fine suspension of the active substance in aphysiologically acceptable aqueous or non-aqueous solvent and areusually presented in single or multidose quantities in sterile form in asealed container, which can take the form of a cartridge or refill foruse with an atomizing device. Alternatively, the sealed container may bea unitary dispensing device, such as a single dose nasal inhaler or anaerosol dispenser fitted with a metering valve which is intended fordisposal after use. Where the dosage form comprises an aerosoldispenser, it will contain a propellant, which can be a compressed gas,such as compressed air or an organic propellant, such asfluorochlorohydrocarbon. The aerosol dosage forms can also take the formof a pump-atomizer.

Buccal or Sublingual Administration

Compositions suitable for buccal or sublingual administration includetablets, lozenges, and pastilles, where the active ingredient isformulated with a carrier, such as sugar, acacia, tragacanth, or gelatinand glycerine. Compositions for rectal administration are convenientlyin the form of suppositories containing a conventional suppository base,such as cocoa butter.

The compounds of the invention may be administered to an animal alone orin combination with pharmaceutically acceptable carriers, as notedabove, the proportion of which is determined by the solubility andchemical nature of the compound, chosen route of administration, andstandard pharmaceutical practice.

Dosage Amounts

The amount of active ingredient in the compositions of the invention canbe varied. One skilled in the art will appreciate that the exactindividual dosages may be adjusted somewhat depending upon a variety offactors, including the protein being administered, the time ofadministration, the route of administration, the nature of theformulation, the rate of excretion, the nature of the subject'sconditions, and the age, weight, health, and gender of the patient.Generally, dosage levels of between 0.1 μg/kg to 100 mg/kg of bodyweight are administered daily as a single dose or divided into multipledoses. Desirably, the general dosage range is between 250 μg/kg to 5.0mg/kg of body weight per day. Wide variations in the needed dosage areto be expected in view of the differing efficiencies of the variousroutes of administration. For instance, oral administration generallywould be expected to require higher dosage levels than administration byintravenous injection. Variations in these dosage levels can be adjustedusing standard empirical routines for optimization, which are well knownin the art. In general, the precise therapeutically effective dosagewill be determined by the attending physician in consideration of theabove identified factors.

Therapeutic Uses and Screening Methods

Compounds disclosed herein can be used to treat disorders wherenecroptosis is likely to play a substantial role (e.g., cerebralischemia, traumatic brain injury, and other disorders described herein).Compounds of the invention can also be used in screening methods toidentify targets of necroptosis and to identify additional inhibitors ofnecroptosis, as well as in assay development.

Compounds disclosed herein can be evaluated for their pharmacologicalproperties in animal models of disease. The compounds identified todecrease necrosis or necroptosis may be structurally modified andsubsequently used to decrease necrosis or necroptosis, or to treat asubject with a condition in which necrosis or necroptosis occurs. Themethods used to generate structural derivatives of the small moleculesthat decrease necrosis or necroptosis are readily known to those skilledin the fields of organic and medicinal chemistry.

Therapy according to the invention may be performed alone or inconjunction with another therapy, for example in combination withapoptosis inhibitors, and may be provided at home, the doctor's office,a clinic, a hospital's outpatient department, or a hospital. Treatmentgenerally begins at a hospital so that the doctor can observe thetherapy's effects closely and make any adjustments that are needed. Theduration of the therapy depends on the age and condition of the patient,as well as how the patient responds to the treatment. Additionally, aperson having a greater risk of developing a condition may receiveprophylactic treatment to inhibit or delay symptoms of the disease.

In some embodiments, the compounds and methods of the invention can beused to treat any of the following disorders where necroptosis is likelyto play a substantial role: a neurodegenerative disease of the centralor peripheral nervous system, the result of retinal neuronal cell death,the result of cell death of cardiac muscle, the result of cell death ofcells of the immune system; stroke, liver disease, pancreatic disease,the result of cell death associated with renal failure; heart,mesenteric, retinal, hepatic or brain ischemic injury, ischemic injuryduring organ storage, head trauma, septic shock, coronary heart disease,cardiomyopathy, bone avascular necrosis, sickle cell disease, musclewasting, gastrointestinal disease, tuberculosis, diabetes, alteration ofblood vessels, muscular dystrophy, graft-versus-host disease, viralinfection, Crohn's disease, ulcerative colitis, asthma, and anycondition in which alteration in cell proliferation, differentiation orintracellular signaling is a causative factor.

Disease states that may be treated by the methods according to theinvention, include any disease state that is characterized bynecroptosis, including, without limitation, organ ischemia (e.g.,stroke, myocardial infarction and retinal ischemia), traumatic braininjury, liver injury, cancer chemo/radiation therapy-induced necrosis,acute necrotizing pancreatitis and forms of neurodegeneration.

Conditions Caused by Alteration in Cell Proliferation, Differentiation,or Intracellular Signalling

Conditions in which alteration in cell proliferation, differentiation orintracellular signaling is a causative factor include cancer andinfection, e.g., by viruses (e.g., acute, latent and persistent),bacteria, fungi, or other microbes. Exemplary viruses are humanimmunodeficiency virus (HIV), Epstein-Barr virus (EBV), cytomegalovirus(CMV)5 human herpesviruses (HHV), herpes simplex viruses (HSV), humanT-Cell leukemia viruses (HTLV)5 Varicella-Zoster virus (VZV), measlesvirus, papovaviruses (JC and BK), hepatitis viruses, adenovirus,parvoviruses, and human papillomaviruses.

Neurodegenerative Diseases

Exemplary neurodegenerative diseases are Alzheimer's disease,Huntington's disease, Parkinson's disease, amyotrophic lateralsclerosis, HIV-associated dementia, cerebral ischemia, amyotropiclateral sclerosis, multiple sclerosis, Lewy body disease, Menke'sdisease, Wilson's disease, Creutzfeldt-Jakob disease, and Fahr disease.Exemplary muscular dystrophies or related diseases are Becker's musculardystrophy, Duchenne muscular dystrophy, myotonic dystrophy, limb-girdlemuscular dystrophy, Landouzy-Dejerine muscular dystrophy,facioscapulohumeral muscular dystrophy (Steinert's disease), myotoniacongenita, Thomsen's disease, and Pompe's disease. Muscle wasting can beassociated with cancer, AIDS, congestive heart failure, and chronicobstructive pulmonary disease, as well as include necrotizing myopathyof intensive care.

Compounds and methods of the invention can additionally be used to boostthe immune system, whether or not the patient being treated has animmunocompromising condition. For example, the compounds describedherein can be used in a method to strengthen the immune system duringimmunization, e.g., by functioning as an adjuvant, or by being combinedwith an adjuvant.

Kits

Any of the compounds or pharmaceutical compositions of the invention canbe used together with a set of instructions, i.e., to form a kit. Thekit may include instructions for use of the compounds of the inventionin a screening method or as a therapy as described herein.

The following non-limiting examples are illustrative of the presentinvention.

EXAMPLES Example 1 Preparation of [1,2,3]thiadiazole derivatives ofFormula (I-a)

The [1,2,3]thiadiazole derivatives are prepared according to the methodoutlined in Scheme 1. Meldrum's acid was treated with acyl chlorides inthe presence of pyridine to give β-ketoester (step (a); Oikawa et al.,J. Org. Chem. 43: 2087 (1978)). The esters were allowed to react withmono-Boc-hydrazine in the presence of a catalytic amount ofp-toluenesulfonic acid (p-TsOH) to give the corresponding imines (step(b); Thomas et al., J. Med. Chem. 28: 442(1985)). Cyclization in thepresence of thionyl chloride yielded the [1,2,3]thiadiazole esters (step(c)). Base hydrolysis of the esters provided acids (step (d)). Thesematerials were coupled with various amines utilizing HBTU (Method A),the corresponding acyl chlorides (Method B) or through the use of EDCI(Method C) to give amides of Formula (I-b).

Example 2 Preparation of Compounds (13) and (16)

Compound 13 was prepared according to the procedure outlined in Scheme2. The ester was reduced with sodium borohydride (step (a)) and theproduct alcohol was converted to the corresponding aldehyde utilizingDess-Martin reagent (step (b)). The aldehyde was condensed with2-chloro-6-fluorobenzylamine in the presence of anhydrous magnesiumsulfate to give an imine, which was subsequently reduced with sodiumtriacetoxyborohydride to give the secondary amine 13 (step (c)). Theimide derivative 16 was also prepared starting with a carboxylic acidwhich was first converted to the corresponding acid chloride (step (d)).This material was then allowed to react with the anion of2-chloro-6-fluorobenzamide generated with sodium hydride to give imide16 in 34% yield (step (e)).

Example 3 Preparation of α-substituted (±)-2-chloro-6-fluorobenzylamines

The α-substituted (±)-2-chloro-6-fluorobenzylamines were preparedaccording to Scheme 3 (Polniaszek et al., J. Org. Chem., 55: 215(1990)).2-Chloro-6-fluorophenyl ketones were reduced with borane-tetrahydrofurancomplex to give the secondary alcohols (step (a)). The alcohols wereconverted to the corresponding phthalimides via a Mitsonobu reaction(step (b)). The benzylamines were isolated following treatment withhydrazine monohydrate (step (c)).(S)-1-(2-Chloro-6-fluorophenyl)ethylamine was prepared by treating thebenzonitrile starting material with methyl magnesium chloride followedby treatment with acetic anhydride to give α-enamide (step (d)).Asymmetric hydrogenation in the presence of the chiral catalyst (S,S)-Me-BPE-Rh gave the corresponding amide (step (e); Burk et al., J. Am.Chem. Soc. 118: 5142(1996)). Acid hydrolysis of the amide yielded theoptically pure amine (step (f)), isolated as the hydrochloride salt.

Example 4 Synthesis of 3-Alkyl pyrrole derivatives (Compounds (81),(82), and (83))

3-Alkyl pyrrole derivatives were prepared according to the procedureoutlined in Scheme 1. Glycine ethyl ester, 5, was treated withp-toluenesulfonyl chloride (Ts-Cl) to give 6, which upon treatment with4-diethylaminobutan-2-one in the presence of t-BuOK gave 7. Dehydrationwith POCl₃ yielded the dihydropyrrole derivative 8. Elimination in thepresence of sodium ethoxide generated pyrrole derivative 9. The pyrrolenitrogen was deprotonated with sodium hydride and alkylated to give 10.The ester was hydrolyzed with aqueous KOH in MeOH and then thecorresponding acid 11 was converted to amides (81), (82), and (83) usingEDCI.

Example 5 Synthesis of 1-Alkyl pyrrole derivatives

1-Alkyl pyrrole derivatives were prepared according to the procedureoutlined in Scheme 2. Methyl 2-pyrrolecarboxylate, 100, was deprotonatedusing sodium hydride and then alkylated to give 101. The ester washydrolyzed to give acid 102, which was coupled to a2-chloro-6-fluorobenzylamine utilizing EDCI to give amide 103.Regioselective bromination with NBS gave 104. Finally, conversion of thearyl bromide to a nitrile was accomplished utilizing apalladium-mediated coupling with zinc cyanide to give 105 in excellentyield.

Example 6 Synthesis of cyano- and halo-substituted pyrrole derivatives

Cyano- and halo-substituted pyrrole derivatives were prepared accordingto the procedure outlined in Scheme 3. 1-Alkylpyrroles 101 were allowedto react with chlorosulfonyl isocyanate to give two readily separableregioisomeric cyanopyrrole derivatives 106 and 107 (1:4). Each wasconverted the corresponding acid and then coupled with2-chloro-6-fluorobenzylamine to give 108 and 109, respectively. Methyl2-pyrrolecarboxylate, 100, was also regioselectively chlorinated witht-butyl hypochlorite to give 110. N-alkylation gave 111 and subsequentester hydrolysis yielded 112, which was coupled with2-chloro-6-fluorobenzylamine to give compound (78).

Example 7 Evaluation of Necroptosis Inhibitory Activity by Thiadiazoles

Evaluation of necroptosis inhibitory activity was performed using aFADD-deficient variant of human Jurkat T cells treated with TNF-α aspreviously described (Degterev et al., Nat. Chem. Biol. 1:112 (2005) andJagtap et al., J. Med. Chem. 50: 1886 (2007)). Utilizing theseconditions the cells efficiently underwent necroptosis, which wascompletely and selectively inhibited by 1 (see Scheme 7; EC₅₀=0.050 μM).For EC₅₀ value determinations, cells were treated with 10 ng/mL of humanTNF-α in the presence of increasing concentration of test compounds for24 hours followed by ATP-based viability assessment.

ATP-based viability assessment: Briefly, necroptosis activity wasperformed using a FADD-deficient variant of human Jurkat T cells treatedwith TNF-α. For EC₅₀ value determinations, cells (500,000 cells/mL, 100μL per well in a 96-well plate) were treated with 10 ng/mL of humanTNF-α in the presence of increasing concentration of test compounds for24 hours at 37° C. in a humidified incubator with 5% CO₂ followed byATP-based viability assessment. Stock solutions (30 mM) in DMSO wereinitially prepared and then diluted with DMSO to give testing solutions,which were added to each test well. The final DMSO concentration was0.5%. Eleven compound test concentrations (0.030-100 μM) were used. Eachconcentration was done in duplicate.

Cell viability assessments were performed using a commercial luminescentATP-based assay kit (CellTiter-Glo, Promega, Madison, Wis.) according tothe manufacturer's instructions. Briefly, 40 μL of the cell lysis/ATPdetection reagent was added to each well. Plates were incubated on arocking platform for 10 minutes at room temperature and luminescence wasmeasured using a Wallac Victor 3 plate-reader (Perkin Elmer, Wellesley,Mass.). Cell viability was expressed as a ratio of the signal in thewell treated with TNF-α and compound to the signal in the well treatedwith compound alone. This was done to account for nonspecific toxicity,which in most cases was <10%. EC₅₀ values were calculated usingnonlinear regression analysis of sigmoid dose-response (variable slope)curves from plots of log[I] verses viability values.

Table 2 provides EC₅₀ determinations of necroptosis inhibition inFADD-deficient Jurkat T cells treated with TNF-α by compounds of theinvention having formula (I-a). The standard deviation is <10%.

TABLE 2 (I-a)

Compound R¹ R² R³ EC₅₀ (μM) 25 Me H 2-Cl-6-F—Ph 1.0 26 Me Me 2-Cl-6-F Ph11 27 Me H 2,6-di-F Ph 3.5 28 Me H 2-Me Ph 27 29 Me H 2-OMe Ph >100 30n-Pr H 2-Cl-6-F Ph 4.1 31 i-Pr H 2-Cl-6-F Ph 0.58 32 c-Pr H 2-Cl-6-F Ph0.50 33 c-Bu H 2-Cl-6-F Ph 0.60 34 c-Pentyl H 2-Cl-6-F Ph 1.9 35 c-Hex H2-Cl-6-F Ph 6.0 36 t-Bu H 2-Cl-6-F Ph 18 37 Ph H 2-Cl-6-F Ph >100 38c-Pr H 2-Cl-6-F Ph 6.0 39 c-Pr H 2,6-di-F Ph 1.5 40 c-Pr H 2-F Ph 1.5 41c-Pr H 2-Cl-6-Me Ph 10 42 c-Pr H 2-Cl-6-(OPh) Ph >100 43 c-Pr H2-Cl-6-CN Ph >100 44 c-Pr H 2-F-6-CF₃ Ph >100 45 c-Pr H 1-naphthyl >10046 c-Pr H 2-Py 40 47 c-Pr H 3-F-2-Py 9.6 48 c-Pr H 2-Cl-3,3-di-F Ph 0.5249 c-Pr H 2-Cl-2,6-di-F Ph 0.18 50 c-Pr Me 2-Cl-6-F Ph 16 c-Pr =cyclopropyl; c-Bu = cyclobutyl; c-Hex = cyclohexyl; Py = pyridyl

The initial [1,2,3]thiadiazole necroptosis inhibitor discovered duringhigh throughput screening (HTS) was (25), with an EC50=1 μM (Table 2).Altering the amide NH, for example, through simple methylation (26 vs 25and 50 vs 32), resulted in significant loss of activity. Introduction ofbranching into the alkyl group at the 4-position of the[1,2,3]thiadiazole increased activity, with i-Pr (31), c-Pr (32) andc-Bu (33) showing the best results. However, introduction of a t-Bu (36)or phenyl (37) at this position resulted in loss of activity. The2-chloro-6-fluoro substitution of the phenyl ring also appeared to benecessary for potent activity. For example, compounds with a2-methylphenyl (28) or 2-methoxyphenyl (29) were less active. Inaddition, the 2,6-dichloro (38) or 2,6-difluoro (39) substitutedderivatives were also less active compared to the 2-chloro-6-fluorosubstitution (32). Consistent with these findings, removing one of thehalogens (40) or replacing one of the halogens with small (41) or large(42) electron donating groups also resulted in decreased activity.Replacing one of the halogens with other electron withdrawing groups,such as cyano (43) or CF3 (44), did not restore activity. Replacing the2-chloro-6-fluorophenyl with a 1-naphthyl (45), 2-pyridyl (46) orsubstituted 2-pyridyl (47) was detrimental to activity. However,addition of a halogen to the 3-position of the 2-chloro-6-fluorophenyl(49) gave an increase in necroptosis inhibition activity with an EC50value of 0.18 μM.

Additional changes to the linker between the [1,2,3]thiadiazole and the2-chloro-6-fluorophenyl were examined. Table 3 provides the EC₅₀determinations of necroptosis inhibition in FADD-deficient Jurkat Tcells treated with TNF-α by compounds having Formula (I-b). Thecorresponding secondary amine (13) and imide (16) derivatives of 32 wereinactive. Also, the benzylamide was necessary, with the homologousphenethyl amide (51) and the truncated anilide (52) being significantlyless active. Compound 52 was prepared in low yield (10%) by allowing 15to react with 2,6-difluoroaniline in THF and pyridine at roomtemperature. The reaction was unsuccessful with 2-chloro-6-fluoroanilinepresumably due to steric hindrance. Introduction of a methyl group (53)onto the benzylic position gave a slight increase in activity. Quitesurprisingly, when the two enantiomers of 53 were examined, all of thenecroptosis activity resided in the (S)-enantiomer (55). However,increasing the steric bulk of the benzylic substituent to n-Bu (56),phenyl (57) or gem-dimethyl (58) resulted in loss of activity.

TABLE 3 (I-b)

Compound X Y R (R)/(S) EC₅₀ (μM)^(a) 13 CH₂ CH₂ Cl — >100 16 C═O C═O Cl— >100 51 C═O CH₂CH₂ Cl — 27 52 C═O — F — >100 53 C═O CH(Me) Cl (R)/(S)0.40 54 C═O CH(Me) Cl (R) >100 55 C═O CH(Me) Cl (S) 0.28 56 C═O CH(n-Bu)Cl (R)/(S) >100 57 C═O CH(Ph) Cl (R)/(S) >100 58 C═O C(Me)₂ Cl — >100

Finally, the [1,2,3]thiadiazole was examined. Table 4 shows the EC₅₀determinations of necroptosis inhibition in FADD-deficient Jurkat Tcells treated with TNF-α using compounds having the Formula (I-f).Replacement with a variety of thiazoles (59-61) or an oxazole (62) wasdetrimental to activity. Likewise, the pyridazine (63), which attemptedto replace the sulfur of the [1,2,3]thiadiazole with a —CH═CH—, was alsoinactive. However, moderate activity could be obtained with a variety ofthiophene derivatives (64-74), except for the ethoxy derivative 75 andthe sulfone derivative 76. In two case (66 and 74) the necroptosisactivity approached that seen for the most potent [1,2,3]thiadiazoles.However, replacement of the [1,2,3]thiadiazole with a furan (77) wasless effective.

TABLE 4 (I-f)

Compound X₃ X₂ X₁ R¹ R⁷ EC₅₀ (μM)^(a) 59 S CH N Me H 20 60 S CHMe N MeH >100 61 S CH-(4-ClPh) N Me H >100 62 O CH N Me H >100 63 CH═CH N N MeH >100 64 S CH CH Me H 7.0 65 S CH CH Me Me 3.9 66 S CH CBr Me H 0.75 67S CH CCN Me H 1.2 68 S CH CH c-Pr H 5.1 69 S CH CMe Cl H 3.9 70 S CH CHCl H 9.6 71 S CMe CH H H 3.9 72 S CH CH H H 9.4 73 S CH CMe H H 3.7 74 SCH C(CH₂)₄ H 0.48 75 S CH CH OEt H >100 76 S CH CR⁴ Me H >100 77 O CH CHMe H 13 Note: R⁴ in Compound (76) is SO₂(4-chlorophenyl)

Example 8 Evaluation of Pyrrole Compounds

Evaluation of compounds 78-99 (Table 5) for necroptosis inhibitoryactivity was also performed. For EC₅₀ value determinations, cells weretreated with 10 ng/mL of human TNF-α in the presence of increasingconcentration of test compounds for 24 hours followed by ATP-basedviability assessment.

Microsome stability was determined in pooled mouse liver microsomes.Test compound (3 μM final concentration) along with 0.5 mg/mL microsomeprotein and 1 mM NADPH was incubated for 0, 5, 15, 30 and 60 minutes.Incubation of test compound and microsomes in the absence of NADPHserved as a negative control. The samples were quenched with methanoland centrifuged for 20 minutes at 2500 rpm to precipitate proteins.Sample supernatants were analyzed (N=3) by LC/MS. The In peak area ratio(compound peak area/internal standard peak area) was plotted againsttime and the slope of the line determined to give the elimination rateconstant [k=(−1)(slope)]. The half life (t_(1/2) in minutes), and the invitro intrinsic clearance (CL_(int) in μL/min/mg protein) werecalculated according to the following equations, where V=incubationvolume in μL/mg protein:

${t_{1/2} = \frac{0.693}{k}};{{CL}_{int} = {\frac{V(0.693)}{t_{1/2}}.}}$

The results of the biological studies are shown in Table 5.

TABLE 5 (I-d)

Compound EC₅₀ (μM)a T_(1/2) (minutes) CL_(int) (μL/minutes/mg protein)78 0.74 15.4 89.8 ± 3.0 79 >20 — — 80 >20 — — 81 4.9 — — 82 >20 — — 837.8 — — 84 0.90 17.2 80.6 ± 4.4 85 0.44 — — 86 >20 — — 87 >20 — — 88 >20— — 89 2.1 — — 90 0.52 — — 91 >20 — — 92 >20 — — 93 2.4 — — 94 0.34 42.332.8 ± 2.2 95 1.9 — — 96 1.4 — — 97 0.092 236   5.9 ± 2.5 98 >20 — —99 >20 — —

Example 9 Determination of “Universal” and Diverse Celltype/Stimulus-Specific Necroptosis Inhibitors

The compounds described herein may show universal activity in a broadrange of necroptosis cellular systems or activity may be restricted tospecific cell types/stimuli. The compounds described herein are expectedto offer advantages, for example, under conditions where moleculespecificity may be beneficial, such as treating chronic conditions likeneurodegenerative diseases.

Activity may be demonstrated using the procedures known in the art (see,for example, Teng et al., Bioorg. Med. Chem. Lett., 15: 5039 (2005) andJagtap et al., J. Med. Chem. 50: 1886(2007)). We performed similaranalyses with the [1,2,3]thiadiazole series. Compound 55 showed the sameactivity profile as necrostatins 1 and 2 (see Scheme 7 for structures)in Jurkat or L929 cells using TNF-α as the necroptosis stimuli (FIG. 1).In addition, 55 is fully active in mouse adult lung fibroblastsstimulated to undergo necroptosis with a combination of TNF-α andzVAD.fmk.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each independent publication or patent application was specificallyand individually indicated to be incorporated by reference.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosure that come within known or customary practice withinthe art to which the invention pertains and may be applied to theessential features hereinbefore set forth, and follows in the scope ofthe claims.

1. A method of treating necrosis or necroptosis in a subject having acondition selected from stroke, myocardial infarction, organ ischemia,traumatic brain injury, liver disease, cancer chemo/radiationtherapy-induced necrosis, acute necrotizing pancreatitis, or aneurodegenerative disease, said method comprising the step ofadministering a compound according to the following formula,

wherein R¹ is a lower alkyl group; R² is selected from hydrogen and anelectron withdrawing group; R³ is selected from hydrogen and an(S)-lower alkyl group; each of R⁴, R⁵, and R⁶ is independently selectedfrom hydrogen, lower alkyl, halogen, amino, amido, alkoxy, and cyano;and each X¹ and X² is, independently, halogen; or any pharmaceuticallyacceptable salt thereof, or stereoisomer thereof.
 2. The method of claim1, wherein said condition is a neurodegenerative disease.
 3. The methodof claim 1, wherein said condition is stroke, liver disease, myocardialinfarction, or retinal ischemia.
 4. The method of claim 1, wherein R¹ isselected from methyl, ethyl, propyl, and butyl; R² is selected fromhydrogen, halogen and cyano; and R³ is selected from hydrogen,(S)-methyl, (S)-ethyl, (S)-propyl, and (S)-butyl.
 5. The method of claim4, wherein R¹ is selected from methyl and isopropyl; R² is selected fromhydrogen, chlorine, bromine and cyano; R³ is selected from hydrogen and(S)-methyl; and each X¹ and X² is independently chlorine or fluorine. 6.The method of claim 5, wherein R¹ is methyl, R² is cyano, R³ is(S)-methyl, X¹ is chlorine, and X² is fluorine.
 7. A method of treatingnecrosis or necroptosis in a subject having a condition selected fromorgan ischemia, traumatic brain injury, liver disease, cancerchemo/radiation therapy-induced necrosis, acute necrotizingpancreatitis, or a neurodegenerative disease, said method comprising thestep of administering a compound selected from the group consisting of:

or any pharmaceutically acceptable salt thereof, or stereoisomerthereof, to said subject in a dosage sufficient to decrease necroptosisor said necrosis.
 8. The method of claim 7, wherein said compound is

or any pharmaceutically acceptable salt thereof, or stereoisomerthereof.
 9. The method of claim 7, wherein said condition is aneurodegenerative disease.
 10. The method of claim 7, wherein saidcondition is stroke, liver disease, myocardial infarction, or retinalischemia.